Induction of secondary axis in hydra revisited: New insights into pattern formation.

In 1909, several years before the famous `Organizer' experiments of Spemann and Mangold, Ethel Browne demonstrated induction of a secondary axis in hydra by grafting a hypostome. Based on this and subsequent work, in the late sixties, Lewis Wolpert proposed the theory of morphogen gradients and positional information. We have studied secondary axis induction by hypostome and foot tissue using three species of hydra as well as transgenic, GFP-expressing lines of hydra. We have found that pieces of hypostome and complete foot of a donor hydra can induce a secondary axis all along (in upper, middle or lower parts of) the body column of a host hydra, both within and across species with comparable rates. Thus, contrary to the available literature, our results show that the host hypostome does not completely inhibit the induction of a secondary axis. The length of the induced axis though is determined by the position of the graft. By using GFP-expressing lines of hydra we have demonstrated that host ectodermal and endodermal cells actively contribute to the secondary axis. On comparison, the hypostome was found to be a stronger and dominant Organizer than the foot. Foot grafting experiments show a transient increase in the host length as well as the distance between the two Organizers. The length becomes normal once the grafted foot reaches the budding zone. Our work brings out several new aspects of the role of positional cues in pattern formation in hydra that can be now be explored at cellular and molecular levels.

Hydra, a common freshwater Cnidarian (family Hydridae, class Hydrozoa), was first described by the Swiss naturalist Abraham Trembley in 1744 and classified by Carl Linnaeus in 1758. The body of a hydra, with a single oralaboral axis, consists of a hypostome surrounded by tentacles, body column, bud(s) and foot (sometimes with peduncle) along it. This axis is called the primary axis and any deviation in this axis due to formation of a new head or foot structure results in the formation of a secondary axis. Using the lateral grafting technique, it has shown that the hypostome, peduncle and basal disc (or foot) could induce a secondary axis when grafted onto the host of the same species (homoplastic transplantation) (1)(2)(3). The head organizer located in the hypostome and two morphogenetic gradients, head activator (HA) and head inhibitor (HI) gradients, that run the length of the oral-aboral axis play essential roles in axis formation of hydra (4,5). A second organizer is known to reside in the foot. Lateral transplants of the two different organizer tissues, when transplanted together onto the middle part of host, antagonized each other's inductive ability (6). A piece of hypostome could induce secondary axis when transplanted within the species (7). By using a labeled transplant and an unlabeled host, it has been shown that cells from the host migrate into the secondary axis and support its growth (7). A full understanding of the underlying mechanisms of axis induction in hydra, however, remains elusive.
We have reexamined the various aspects of secondary axis induction in hydra with a view to further exploring hydra as a model system to study pattern formation. In the present study, we have attempted to do the following: 1. Compare the inductive abilities of hypostome and foot, 2.
Study the interaction between the hypostome and foot Organizers, 3. Study the influence of host hypostome on induction of secondary axis, 4. Compare secondary axis induction within and between species of hydra and 5. Study the contribution of host cells to secondary axis formation.
We have found that when either a piece of hypostome or a foot is grafted on different parts of the host body column within or across species, a secondary axis is induced, the length of which varies depending upon the position of grafting. The inducing capacity of hypostomal tissue is much greater than foot tissue since only a small piece of hypostome induces a second axis while the whole foot is required for induction to occur. The hypostomal and foot organizers seem to actively interact with each other as evident from the active away migration of foot grafted near host hypostome. This migration continues till the new axis reaches the budding zone or just below it, presumably when normal positional information gradients are reestablished. We have also found, contrary to earlier reports, that a secondary axis is induced even in the presence of the host hypostome though the length of the induced axis is drastically reduced. Finally, by using a combination of nontransgenic grafts and transgenic hosts, we confirm that the host cells (both ectodermal and endodermal) contribute to the secondary axis.

Hydra culturing and maintenance
Polyps were cultured at 18°C. The animals were fed with freshly hatched Artemia nauplii and the medium was changed daily. Animals starved for 24 hrs were used in all experiments.
Transplantation experiments were carried out by using Hydra vulgaris Ind-Pune (8)  To determine the role of host hypostome on secondary axis, we used hosts either with or without intact hypostome (Fig.1). A piece of hypostome or foot was isolated from the donor by transverse cut made with a fine needle. An incision was made in the host ectoderm and transplant was inserted into the slit in such a way that a part of it protrudes out from the host. The host was left undisturbed for 2 to

Induction capacity of hypostome within the species (homoplastic transplantation)
The first indication of induction of a secondary axis was a localized swelling of the body wall that became evident within 2 to 3 hrs of grafting. In all, the three species used, namely, H.
vulgaris, homoplastic transplantation resulted in induction of a secondary axis in over 90% of the cases (Table 1). About 5% of grafts apparently got assimilated which may be due to accidental insertion of transplants into the digestive tract of the host.  secondary axis was observed (Fig.2B). On grafting of the piece of hypostome onto the lower body part, a complete secondary axis with foot, body column, hypostome and tentacles was formed (Fig.2C).
Thus, the host hypostome appears to exert an inhibitory effect on the induction of the secondary axis. However, not even a single case did we observe complete inhibition of induction due to host hypostome. After few days, the secondary axis stopped growing and started feeding independently.
None of the grafts detached from the hosts up to ten days. The length of the secondary axis depends on the position of the graft, in that very small, moderate and complete axis was found to be formed in upper, middle and lower part of the body column, respectively. This indicates the presence of a head inhibitor (HI) as reported earlier (10,11). To confirm such an influence of the host hypostome, homplastic transplantations were carried out in hosts with their hypostomes completely removed. It was observed that secondary axis was induced in over 90% of the cases ( Table 2).   Heteroplastic grafting performed after removing host hypostome also resulted in comparable inductions (Table 4). with a sticky foot at the distal end (Fig.3A).

Induction capacity of foot within the species
Interestingly, the distance between host hypostome and induction caused by foot was found to increase till about day 4 post-grafting followed by a reduction that resulted in attaining the normal length by day 6 post-grafting (Fig.4). This phenomenon has appeared to have resulted from active repulsion between the two organizing centers residing in the hypostome of the host and the grafted foot. When foot was grafted in the middle part of the body column, induction having small body column and foot at the distal end has been observed (Fig.3B). Again the distance between host hypostome and new axis was found to increase on  Removal of host hypostome did not influence the induction of secondary axis (Tables 5,6).

Induction capacity of foot across the species
Foot and peduncle of hydra have induction capacity within the species in that they induce proximal secondary axis (foot at the end of new axis) (6). By transplanting the foot of H. magnipapillata to Hydra sp. India and vice versa, we assayed its induction capacity.
When the induction capacity of homospecific and heterospecific grafts was compared, no significant difference was found (Tables 7,8) the nature of the secondary axis was same in both cases.

Host supports the growth of secondary axis
Hypostome has the characteristics of an organizer to induce host tissue to form most of the second axis. In contrast, tissue of the body column has a self-organizing capacity (it divides itself to form an axis) (7). By using stained transplant

Multiple inductions
In order to test whether multiple axes can be induced or not, we performed three intraspecific (H.  The length of hypostome-induced axes differed based on the position of the graft. As observed previously, the increase in total host length as well as distance between host hypostome and grafted foot was also evident here.

Discussion
The hypostome and foot are the two